www.kjurology.org http://dx.doi.org/10.4111/kju.2013.54.9.631 Basic and Translational Research Decreased DBC1 Expression Is Associated With Poor Prognosis in Patients With Non-Muscle-Invasive Bladder Cancer Ui Jae Shim, Il-Seok Lee, Ho Won Kang, Jayoung Kim1,2, Won Tae Kim, Isaac Yi Kim3, Keun Ho Ryu4, Yung Hyun Choi5, Sung-Kwon Moon6, Yong-June Kim, Seok-Joong Yun, Sang-Cheol Lee, Wun-Jae Kim Department of Urology, Chungbuk National University College of Medicine, Cheongju, Korea, 1The Urological Diseases Research Center, Children’s Hospital Boston, Boston, MA, USA, 2Departments of Surgery and Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, USA, 3Section of Urologic Oncology, Cancer Institute of New Jersey, Robert Wood Johnson Medical School, New Brunswick, NJ, USA, 4Database/Bioinformatics Laboratory, Chungbuk National University, Cheongju, Korea, 5Department of Biochemistry, Dongeui University College of Oriental Medicine, Busan, Korea, 6Department of Food and Biotechnology, Chungang University, Seoul, Korea Purpose: The deleted in bladder cancer 1 (DBC1) gene is located within chromosome Article History: 9 (9q32-33), a chromosomal region that frequently shows loss of heterozygosity in blad- received 26 June, 2013 22 July, 2013 der cancer (BC). It is suspected that it acts as a tumor suppressor gene, but its prognostic accepted value remains unclear. The aim of the present study was to investigate the value of DBC1 as a prognostic marker in BC. Materials and Methods: The expression of DBC1 was determined by real-time polymer- ase chain reaction analysis in 344 patients with BC (220 non-muscle-invasive BC [NMIBC] and 124 muscle-invasive BC [MIBC]) and in 34 patients with normal bladder mucosa. The results were compared with clinicopathologic parameters, and the prog- nostic value of DBC1 was evaluated by Kaplan-Meier analysis and a multivariate Cox regression model. Results: DBC1 expression was significantly decreased in patients with MIBC compared with those diagnosed with NMIBC (p=0.010). Patients with aggressive tumor charac- teristics had lower DBC1 expression levels in NMIBC (each, p＜0.05). By multivariate Cox regression analysis, low DBC1 expression was a predictor of progression to MIBC (hazard ratio, 7.104; p=0.013). Kaplan-Meier estimates revealed a significant differ- Corresponding Author: ence in tumor recurrence, progression to MIBC, and cancer-specific survival depending Won Tae Kim on the level of DBC1 expression in NMIBC (log-rank test, each, p＜0.05). Department of Urology, Institute Conclusions: The expression of DBC1 was associated with tumor aggressiveness, pro- for Tumor Research, Chungbuk National University College of gression to MIBC, and survival in NMIBC. Our results suggest that DBC1 expression Medicine, 52 Naesudong-ro, can be a useful prognostic marker for patients with NMIBC. Heungdeok-gu, Cheongju 361-804, Korea Keywords: Bladder cancer; Human DBC1 protein; Prognosis TEL: +82-43-269-6371 FAX: +82-43-269-6144 This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, E-mail: wtkimuro@chungbuk. distribution, and reproduction in any medium, provided the original work is properly cited. ac.kr INTRODUCTION 75% of patients present with non-muscle-invasive bladder cancer (NMIBC), which can be managed with a combina- Bladder cancer is the second most common genitourinary tion of transurethral resection (TUR) and intravesical tumor in human populations, and it was estimated in 2013 therapy [2,3]. However, after TUR about three-quarters of that 72,570 new cases of cancer of the urinary bladder were patients experience tumor recurrence within 2 years, and diagnosed in the United States and 15,210 deaths were at- 20% to 30% of them experience progression to muscle-in- tributable to bladder cancer [1]. At the initial diagnosis, vasive bladder cancer (MIBC) [4,5]. Only 20% of bladder Korean Journal of Urology Ⓒ The Korean Urological Association, 2013 631 Korean J Urol 2013;54:631-637 632 Shim et al cancer patients are diagnosed with MIBC, which causes indicator for bladder cancer. most cancer-specific deaths. Although radical cystectomy is considered the gold standard for the treatment of pa- MATERIALS AND METHODS tients with localized MIBC, about 50% of these patients de- velop metastases within 2 years after cystectomy and sub- 1. Study population sequently die of the disease [6]. We collected bladder tissue from 344 patients with primary Bladder cancer has diverse biological and functional bladder cancer (220 NMIBC and 124 MIBC) and 34 pa- characteristics. Therefore, it is very difficult for urologists tients with nonmalignant, noninflammatory disease. to estimate the success rate of treatment and to counsel pa- Cases were recruited from patients with bladder cancer tients about prognosis [7]. Prognostic information ob- who had been histologically verified with urothelial carci- tained from conventional histopathological parameters noma at our institution. To reduce confounding factors af- such as tumor stage or grade and lymph node status is insuf fecting the analysis and to delineate a more homogeneous ficient to predict outcome. One limitation of the current use study population, any patients diagnosed with a con- of staging is the fact that tumors of a similar stage and grade comitant carcinoma in situ, or for whom data collection was can have a significantly different biology [8]. Nomograms incomplete, as well as patients with short-term follow-up and prognostic modeling approaches that simultaneously periods (less than 6 months) were excluded. The Ethics incorporate many factors have been developed for assess- Committee of Chungbuk National University approved ing the risk of individual patients [3,9,10]. The limited val- this protocol, and written informed consent was obtained ue of the established prognostic markers is that these re- from each subject. The collection and analyses of all sam- quire analysis of new molecular parameters of interest for ples was approved by the Institutional Review Board of predicting the prognosis of bladder cancer patients in a Chungbuk National University (IRB approval number clinical setting [11]. The specific alterations in gene ex- 2006-01-001), and informed consent was obtained from pression that occur as a result of the cross-talk between var- each subject. ious cellular pathways determine the biological behavior Tumors were staged according to the 2002 TNM classi- of a tumor, including its growth, recurrence, progression, fication and the 1973 World Health Organization grading and metastasis, all of which can influence patient survival system [22]. All diagnoses were confirmed by pathological [7,11-14]. To detect and monitor cancer and determine a analysis of frozen sections from cystectomy and TUR likely prognosis, it is necessary to identify molecular mark- specimens. In the case of NMIBC, a TUR of the tumor was ers of the disease that can be used in a clinic. performed. A second TUR was performed 2 to 4 weeks after The deleted in bladder cancer 1 (DBC1) gene, originally the initial resection when a bladder cancer specimen did named DBCCR1, which represents a candidate tumor sup- not include proper muscle or when a high-grade tumor was pressor gene within a precisely mapped region of loss of het- detected. Patients who had multiple tumors, large tumors erozygosity on 9q33, shows the loss of one allele in 60% of (≥3 cm in diameter), or high-grade NMIBC received one all bladder tumors or transcriptional silencing in approx- cycle of intravesical treatment (bacille Calmette-Guerin imately 50% of bladder cancer cell lines by promoter hyper- [BCG] or mitomycin-C). Response to treatment was as- methylation of the 5’ region of DBC1 containing a CpG is- sessed by cystoscopy and urinary cytology. Patients who land [15-18]. Abnormal methylation or deletion of DBC1 were free of disease within 3 months of the commencement has also been described in other tumors including oral squ- of treatment were assessed every 3 months for the first 2 amous cell carcinoma, non-small-cell lung cancer, and years and every 6 months thereafter [5]. We defined re- acute lymphoblastic leukemia [19,20]. DBC1 contains a currence as a relapse of primary NMIBC with a lower or membrane attack complex/perforin domain, which is a equivalent pathologic stage and progression to invasive membrane-disrupting protein that is involved in pore for- bladder cancer. mation during complement-mediated cell lysis [16]. DBC1 is also known to play a role in cell cycle control. Exogenous 2. RNA extraction and construction of cDNA expression of DBC1 protein in human bladder tumor cell One milliliter of TRIzol (Invitrogen, Carlsbad, CA, USA) lines results in suppression of proliferation, and this was added to control and bladder cancer tissue and the sam- DBC1-mediated growth inhibition is due to an increase in ples were homogenized in a 5-mL glass tube. The homoge- the number of cells in the G1 phase of the cell cycle [16,21]. nate was transferred to a 1.5-mL tube and was mixed with Thus far, there has been no evidence for an equivalent func- 200 μL chloroform. After incubation for 5 minutes at 4oC, tion of DBC1, and clinical evidence is insufficient as to the homogenate was centrifuged for 13 minutes at 13,000× whether the inactivation of the DBC1 tumor suppressor g at 4oC. The upper aqueous phase was transferred to a gene influences genetic susceptibility and the disease clean tube and 500 μL isopropanol was added, followed by course of bladder cancer. incubation for 60 minutes at 4oC. The tube was then centri- In the current study, we compared the expression levels fuged for 8 minutes at 13,000×g and 4oC. Then, the upper of DBC1 between normal and cancer tissue to assess the aqueous phase was removed, mixed with 500 μL of 75% contribution of this gene in bladder carcinogenesis.